An intracellular acid phosphatase (IAP) from Pi-starved (−Pi) tomato (Lycopersicon esculentum) suspension cells has been purified to homogeneity. IAP is a purple acid phosphatase (PAP), as the purified protein was violet in colour (λmax=546 nm) and was insensitive to l-tartrate. PAGE, periodic acid–Schiff staining and peptide mapping demonstrated that the enzyme exists as a 142 kDa heterodimer composed of an equivalent ratio of glycosylated and structurally dissimilar 63 (α-subunit) and 57 kDa (β-subunit) polypeptides. However, the nine N-terminal amino acids of the α- and β-subunits were identical, exhibiting similarity to the deduced N-terminal portions of several putative plant PAPs. Quantification of immunoblots probed with rabbit anti-(tomato acid phosphatase) immune serum revealed that the 4-fold increase in IAP activity due to Pi-deprivation was correlated with similar increases in the amount of antigenic IAP α- and β-subunits. IAP displayed optimal activity at pH 5.1, was activated 150% by 10 mM Mg2+, but was potently inhibited by Zn2+, Cu2+, Fe3+, molybdate, vanadate, fluoride and Pi. Although IAP demonstrated broad substrate selectivity, its specificity constant (Vmax/Km) with phosphoenolpyruvate was >250% greater than that obtained with any other substrate. IAP exhibited significant peroxidase activity, which was optimal at pH 9.0 and insensitive to Mg2+ or molybdate. This IAP is proposed to scavenge Pi from intracellular phosphate esters in −Pi tomato. A possible secondary IAP role in the metabolism of reactive oxygen species is discussed. IAP properties are compared with those of two extracellular PAP isoenzymes that are secreted into the medium of −Pi tomato cells [Bozzo, Raghothama and Plaxton (2002) Eur. J. Biochem. 269, 6278–6286].
Abbreviations used: AP, acid phosphatase; Con A, concanavalin A; DTT, dithiothreitol; IAP, intracellular AP; PAP, purple AP; PEG, poly(ethylene glycol); PEP, phosphoenolpyruvate; +Pi and −Pi, Pi-sufficient and Pi-deficient respectively; ROS, reactive oxygen species; SAP, secreted AP.